Species-specific restriction of apobec3-mediated hypermutation

Abstract

Apobec proteins are a family of cellular cytidine deaminases, among which several members have been shown to have potent antiviral properties. This antiviral activity is associated with the ability to cause hypermutation of retroviral cDNA. However, recent research has indicated that Apobec proteins are also able to inhibit retroviruses by other mechanisms that are independent of their deaminase activity. We have compared the antiviral activities of human and murine Apobec3 (A3) proteins, and we have found that, consistent with previous reports, human immunodeficiency virus (HIV) is able to resist human A3G but is sensitive to murine A3, whereas murine leukemia virus (MLV) is relatively resistant to murine A3 (mA3) but sensitive to human A3G. In contrast to previous studies, we observed that mA3 is packaged efficiently into MLV particles. The C-terminal cytidine deaminase domain (CDD2) is required for packaging of mA3 into MLV particles, and packaging did not depend on the MLV viral RNA. However, mA3 packed into MLV particles failed to cause hypermutation of viral DNA, indicating that its deaminase activity is blocked or inhibited. hA3G also caused significantly less hypermutation of MLV than of HIV DNA. Both mA3 and the splice variant mA3Delta5 exhibited some residual antiviral activity against MLV and caused a reduction in the ability of MLV particles to generate reverse transcription products. These results suggest that MLV has evolved specific mechanisms to block the ability of Apobec proteins to mediate deaminase-dependent hypermutation.

FIG. 1.

Species-specific activities of human and mouse Apobec3 proteins. 293T cells were transfected with 10 μg of MLV and 4 μg of pMIGR or 10 μg of HIV plasmid DNA, as well as various amounts of HA-tagged Apobec plasmids (0, 1.5, or 3 μg) or control vector. At 48 h posttransfection, viral supernatants were used to infect either NIH 3T3 cells (for MLV) or CD4- and CXCR4-expressing HOS cells with a GFP reporter under the control of the HIV long terminal repeat promoter (for HIV). At 24 h postinfection, the level of infectious virus in the supernatant was measured by flow cytometry of the infected cells. Infectivity is displayed as a percentage of that for virus alone. The data shown are the average of two independent experiments. Cellular protein from transfected cells was Western blotted for the presence of Apobec proteins (HA), the cellular loading control HMG, and p30 (MLV) or p24 (HIV).

FIG. 2.

mA3 is packaged efficiently into MLV particles. (A) MLV or HIV particles were produced by transfecting 293T cells with 10 μg of MLV or HIV (vif−) DNA with 3 μg of mA3, mA3Δ5, hA3g, or empty vector. At 48 h posttransfection, transfected cellular protein and purified virus particles were isolated and Western blotted for CA, tubulin, or Apobec proteins (HA). (B) The efficiency of mA3Δ5 incorporation into HIV and MLV was compared by transfecting 293T cells with 10 μg of viral DNA plus 3 μg of mA3Δ5 DNA. At 48 h posttransfection, virus was purified from the supernatant. Purified virus was then analyzed by SDS-PAGE and Coomassie blue stain to compare the relative levels of HIV and MLV and Western blotted for mA3Δ5 (HA) to assess incorporation. (C) MLV particles were produced by transfecting 3T3 cells with 10 μg of MLV alone or with 3 μg of mA3Δ5 or mA3. At 48 h posttransfection, virus was purified from supernatant and then analyzed by Western blotting for CA (p30) or mA3Δ5 and mA3 (HA). (D) MLV or HIV particles were produced by transfecting 293T cells with 10 μg of MLV or HIV DNA with 3 μg of mA3, mA3Δ5, or hA3g. At 48 h posttransfection, purified virus particles were isolated, resuspended, and mixed with Triton X-100. After centrifugation, the detergent-soluble supernatant (S) and the insoluble pellet (P) were Western blotted for CA (p30 or p24) or Apobec proteins (HA).

FIG. 3.

CDD2 but not viral RNA is required for mA3 packaging in MLV. (A) MLV VLPs were generated by transfection of 293T cells with a plasmid that expresses all MLV viral proteins but lacks an RNA packaging element. VLPs were produced in the presence of mA3Δ5 (left panels) or mA3 (right panels) and in the presence or absence of a viral RNA (pMIGR). At 48 h posttransfection, both transfected cells and purified MLV particles were Western blotted for the presence of mA3Δ5 or mA3. (B) 293T cells were transfected with 10 μg of MLV DNA and 3 μg of mA3Δ5, mA3Δ5-CCAA1, or mA3Δ5-CCAA2. At 48 h posttransfection, transfected cells and virus particles purified from the supernatant were Western blotted for mA3Δ5 and p30.

FIG. 4.

MLV blocks Apobec-mediated hypermutation. (A) MLV or HIV (vif−) particles were generated by transfecting 293 cells with 10 μg of viral DNA and 3 μg of mA3, mA3Δ5, or hA3g and used to infect 3T3 cells At 48 h postinfection, cellular DNA was isolated from infected cells, and the viral GFP cassette (566 nucleotides) was amplified by PCR and TA cloned. A pool of 15 to 20 clones (8 to 12 kb) were sequenced for each virus-Apobec combination, and the number of G-to-A mutations was counted. (B) A representative panel of mutations for four clones from each virus-Apobec combination are shown.

FIG. 5.

Accumulation of MLV DNA is reduced by mA3. (A) MLV particles were produced by transfection of 293T cells with 10 μg of MLV DNA alone or in the presence of mA3Δ5, mA3 or hA3G. Viral supernatants were then used to infect NIH 3T3 cells. At 48 h postinfection, cellular DNA was isolated, and the level of viral DNA was measured by real-time PCR. This measurement was normalized to a cellular genomic DNA control. (B) Accumulation of MLV “early” and “late” reverse transcripts was analyzed identically to the procedure describe for panel A except that cellular DNA was isolated at 12 h postinfection.